Background Paucity of T cells in the immune privileged tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is a major reason that PDAC is refractory to immune checkpoint blockade.1 In this study, we show that human PDAC tumors over-express vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, that inhibits effector T cell responses and regulates chemokine receptor expression on activated T cells.2 3 We thus hypothesized that pharmacological inhibition of VIP receptor signaling could enhance anti-tumor responses in PDAC.
Methods VIP levels in plasma were determined via VIP-specific enzyme immunoassay and confirmed with immunohistochemistry (IHC) of tissue sections. VIP receptor (VIP-R) signaling in C57BL/6 immunocompetent murine models of KPC, MT5 or Panc02 pancreatic cancer was inhibited by daily sub-cutaneous treatment with ANT008 or ANT308, two novel VIP-R antagonists with predicted high binding affinities to VIP receptors.4–7 Multiplex IHC or flow cytometry detected frequencies and phenotypes of intra-tumoral T cells across treatment groups.
Results Human PDAC tumors expressed VIP by immunohistochemistry, and PDAC patients had significantly elevated plasma VIP levels when compared to healthy volunteers (p<0.01, figure 1). Inhibiting VIP-R signaling in combination with anti-PD-1 monoclonal antibody (MoAb) synergistically enhanced T-cell dependent anti-tumor responses in murine PDAC resulting in elimination of tumors in up to 30% of the animals and increased intratumoral CD4+ or CD8+ T cell density in orthotopic murine PDAC (figure 2). VIP-R antagonist+anti-PD-1 combination therapy significantly increased intratumoral T cell activation and the proportion of tumor specific CD8+ T cells when compared to control (p<0.01, figure 3–5). Furthermore, tumor-free mice that had been treated with VIP-R antagonist and anti-PD-1 MoAb remained tumor-free upon tumor rechallenge, indicating that combination treatment induced robust immunological memory. Interestingly, anti-PD-1 monotherapy increased expression of CXCR4 on T cells in tumor draining lymph nodes, a chemokine receptor that has been shown to trap T cells in the extracellular tumor matrix. On the other hand, combination therapy with VIP-R antagonists and anti-PD1 MoAb significantly decreased CXCR4 expression and promoted homing of adoptively-transferred GFP+ T cells into the tumors.
Conclusions VIP-R antagonists represent a novel approach to treat PDAC. VIP and VIP-R sequences are highly conserved between humans and mice,8 and human T cells are activated in vitro following treatment with VIP-R antagonists. Thus, we predict comparable anti-tumor activity of the combination of VIP-R antagonist and anti-PD-1 MoAb in human PDAC patients. Further clinical development of this novel concept will require appropriate pre-clinical pharmacokinetic and toxicology studies.
Acknowledgements The authors thank healthy volunteers and patients for blood and/or tissue samples. The authors also thank the shared resources at Emory University, namely the Emory Integrated Genomics Core (EIGC), Emory Flow Cytometry Core (EFCC), Cancer Animal Models Shared Resource (CAMS), Cancer Tissue Pathology Core (CTP), Biostatistics Shared Resource (BSR) and Integrated Cellular Imaging Core (ICI), that provided services or instruments at subsidized cost to conduct some of the reported experiments. BioRender was used to make figure 4A and 5C. This work was supported in part by Katz Foundation funding and Emory School of Medicine Dean's Imagine, Innovate and Impact (I3) venture award to Edmund K. Waller and NIH R01 CA207619 awarded to Susan N. Thomas. Part of the cost for the immunohistochemistry staining of tissues was covered by Winship Cancer Institute Development Discovery and Therapeutic Program Pilot funding to Sruthi Ravindranathan.
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Ethics Approval All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) at Emory University. De-identified blood samples from consented patients with PDAC (IRB 00087397) or healthy volunteers (IRB 00046063) were obtained with approval from Institutional Review Boards.
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